The present invention relates to novel cytotoxic agents and their therapeutic use. More specifically, the invention relates to novel cytotoxic agents comprising modified doxorubicins/daunorubicins and their therapeutic use. These novel cytotoxic agents have therapeutic use as a result of delivering the modified doxorubicins/daunorubicins to a specific cell population in a targeted fashion by chemically linking the doxorubicin/daunorubicin to a cell binding agent.
Many reports have appeared on the attempted specific targeting of tumor cells with monoclonal antibody-drug conjugates (Sela et al, in Immunoconjugates 189-216 (C. Vogel, ed. 1987); Ghose et al, in Targeted Drugs 1-22 (E. Goldberg, ed. 1983); Diener et al, in Antibody mediated delivery systems 1-23 (J. Rodwell, ed. 1988); Pietersz et al, in Antibody mediated delivery systems 25-53 (J. Rodwell, ed. 1988); Bumol et al, in Antibody mediated delivery systems 55-79 (J. Rodwell, ed. 1988). Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, and chlorambucil have been conjugated to a variety of murine monoclonal antibodies. In some cases, the drug molecules were linked to the antibody molecules through an intermediary carrier molecule such as serum albumin (Garnett et al, 46 Cancer Res. 2407-2412 (1986); Ohkawa et al, 23 Cancer Immunol Immunother. 81-86 (1986); Endo et al, 47 Cancer Res. 1076-1080 (1980)), dextran (Hurwitz et al, 2 Appl. Biochem. 25-35 (1980); Manabi et al, 34 Biochem. Pharmacol. 289-291 (1985); Dillman et al, 46 Cancer Res. 4886-4891 (1986); Shoval et al, 85 Proc. Natl. Acad. Sci. 8276-8280 (1988)), or polyglutamic acid (Tsukada et al, 73 J. Natl. Canc. Inst. 721-729 (1984); Kato et al, 27 J. Med. Chem. 1602-1607 (1984); Tsukada et al, 52 Br. J. Cancer 111-116 (1985)).
An array of linker technologies has been employed for the preparation of such immunoconjugates and both cleavable and non-cleavable linkers have been investigated. In most cases, the full cytotoxic potential of the drugs could only be observed, however, if the drug molecules could be released from the conjugates in unmodified form at the target site.
One of the cleavable linkers that has been employed for the preparation of antibody-drug conjugates is an acid-labile linker based on cis-aconitic acid that takes advantage of the acidic environment of different intracellular compartments such as the endosomes encountered during receptor mediated endocytosis and the lysosomes. Shen and Ryser introduced this method for the preparation of conjugates of daunorubicin with macromolecular carriers (102 Biochem. Biophys. Res. Commun. 1048-1054 (1981)). Yang and Reisfeld used the same technique to conjugate daunorubicin to an anti-melanoma antibody (80 J. Natl. Canc. Inst. 1154-1159 (1988)). Dillman et al also used an acid-labile linker in a similar fashion to prepare conjugates of daunorubicin with an anti-T cell antibody (48 Cancer Res. 6097-6102 (1988)).
An alternative approach, explored by Trouet et al, involved linking daunorubicin to an antibody via a peptide spacer arm (79 Proc. Natl. Acad. Sci. 626-629 (1982)). This was done under the premise that free drug could be released from such a conjugate by the action of lysosomal peptidases.
In vitro cytotoxicity tests, however, have revealed that antibody-drug conjugates rarely achieved the same cytotoxic potency as the free, unconjugated drugs. This suggested that mechanisms by which drug molecules are released from the antibodies are very inefficient. In the area of immunotoxins, conjugates formed via disulfide bridges between monoclonal antibodies and catalytically active protein toxins were shown to be more cytotoxic than conjugates containing other linkers. (See, Lambert et al, 260 J. Biol. Chem. 12035-12041 (1985); Lambert et al, in Immunotoxins 175-209 (A. Frankel, ed. 1988); Ghetie et al, 48 Cancer Res. 2610-2617 (1988)). This was attributed to the high intracellular concentration of glutathione contributing to the efficient cleavage of the disulfide bond between an antibody molecule and a toxin. Despite this, there are only a few reported examples of the use of disulfide bridges for the preparation of conjugates between drugs and macromolecules. Shen et al (260 J. Biol. Chem. 10905-10908 (1985)) described the conversion of methotrexate into a mercaptoethylamide derivative followed by conjugation with poly-D-lysine via a disulfide bond. Another report described the preparation of a conjugate of the trisulfide-containing toxic drug calicheamicin with an antibody (Hinman et al, 53 Cancer Res. 3336-3342 (1993)).
One reason for the lack of disulfide linked antibody-drug conjugates is the unavailability of cytotoxic drugs possessing a sulfur atom-containing moiety that can be readily used to link the drug to an antibody via a disulfide bridge. Furthermore, chemical modification of existing drugs is difficult without diminishing their cytotoxic potential.
Another major drawback with existing antibody-drug conjugates is their inability to deliver a sufficient concentration of drug to the target site because of the limited number of targeted antigens and the relatively moderate cytotoxicity of cancerostatic drugs like methotrexate, daunorubicin, doxorubicin and vincristine. In order to achieve significant cytotoxicity, linkage of a large number of drug molecules either directly to the antibody or through a polymeric carrier molecule becomes necessary. However such heavily modified antibodies often display impaired binding to the target antigen and fast in vivo clearance from the blood stream.
In spite of the above described difficulties, useful cytotoxic agents comprising cell binding moieties and the group of cytotoxic drugs known as maytansinoids have been reported (U.S. Pat. Nos. 5,208,020; 5,416,064; and R. V. J. Chari, 31 Advanced Drug Delivery Reviews 89-104 (1998)). Similarly, useful cytotoxic agents comprising cell binding moieties and analogues and derivatives of the potent antitumor antibiotic CC-1065 have also been reported (U.S. Pat. Nos. 5,475,092 and 5,585,499, both of which are expressly incorporated herein by reference).
Doxorubicin (Adriamycin) and daunorubicin (Daunomycin) are cytotoxic natural products that are widely used in the treatment of cancer. These compounds belong to the family of compounds called anthracyclines. Anthracyclines are DNA interacting agents that intercalate into the DNA and interfere with its template function causing cell death. While doxorubicin and daunorubicin are useful agents in the treatment of cancer, their anti-tumor activity is limited because of their non-specific toxicity towards normal cells.
Further, compounds like doxorubicin and daunorubicin themselves are not sufficiently potent to be used in conjugates of cell binding agents. Several attempts to link these compounds to antibodies have resulted in conjugates with low potency and poor target-selectivity (R. S. Greenfield et al, 50 Cancer Res. 6600-6607 (1990); R. S. Greenfield et al, 50 Cancer Res. 6608-6614 (1990); R. V. J. Chari, 31 Advanced Drug Delivery Revs. 89-104 (1998)). Thus, these conjugates have proven ineffective in human clinical trials (A. W. Tolcher et al, 17 J. Clin. Oncol. 478-484 (1999)). A few morpholino analogs with greater potency than either daunorubicin or doxorubicin have been described (E. M. Acton et al, 27 J. Med. Chem. 638-645 (1984); E. M. Acton et al, 29 J. Med. Chem., 1225-1230 (1985); E. M. Acton et al, 29 J. Med. Chem. 2074-2079 (1986); U.S. Pat. Nos. 4,464,529, 4,672,057, 5,304,687(FIG. 1); and recently, a pyrrolinodoxorubicin ((5) in FIG. 1) has been described (A. Nagy et al, 93 Proc. Natl. Acad Sci., 2464-2469 (1996); U.S. Pat. No. 5,843,903). However, these compounds lack a functionality that allows linkage via a cleavable bond to cell binding agents. Also, one attempt to link morpholinodoxorubicin to an antibody via an acid-labile linker led to an unstable conjugate that was inactive (B. M Mueller et al, 1 BioConjugate Chem. 325-330 (1990)).
Accordingly, a method of treating diseases with doxorubicins/daunorubicins wherein their side effects are reduced without compromising their cytotoxicity is greatly needed.
One object of the present invention is to provide modified doxorubicins/daunorubicins that are highly toxic and that can still be effectively used in the treatment of many diseases.
Another object of the present invention is to provide novel modified doxorubicins/daunorubicins.
These and other objects have been achieved by providing a cytotoxic agent comprising one or more modified doxorubicins or daunorubicins linked to a cell binding agent.
In a second embodiment, the present invention provides a therapeutic composition comprising:
(A) a therapeutically effective amount of one or more modified doxorubicins or daunorubicins linked to a cell binding agent, and
(B) a pharmaceutically acceptable carrier.
In a third embodiment, the present invention provides a method of killing selected cell populations comprising contacting target cells or tissue containing target cells with an effective amount of a cytotoxic agent comprising one or more modified doxorubicins or daunorubicins linked to a cell binding agent.
In a fourth embodiment, the present invention provides modified doxorubicins or daunorubicins comprising a linking group capable of linking said modified doxorubicins or daunorubicins to a chemical moiety.